The toxic effects observed during episodes of chemical nerve agent exposure are primarily the result of the irreversible inhibition of acetylcholinesterases (AChEs). These enzymes play an important role in the regulation of neural signalling throughout the body. Inhibition of AChEs invariably leads to the deregulation of post-synaptic targets (i.e. heart, lungs, etc) and may lead to death. Human paraoxonase (HuPONI) is a serum protein capable of hydrolyzing these deadly toxins, however, its catalytic capacity must be optimized before it can be successfully used as a viable antidote to treat nerve agent poisoning. The objective of this application is the design and synthesis of HuPONI variants capable of detoxifying nerve agents before they can reach their physiological targets and exert a lethal effect. To do this we will define amino acid residues (in or near the active site of the enzyme) that may play a role in the breakdown of chemical nerve agents. By using rational design we will generate variants of the native HuPONI enzyme and test their capacity to catalyltically hydrolyze nerve agents such as tabun (GA), sarin (GB), soman, (GD), and VX. This will be accomplished by the examination of the alteration in the affinity of HuPONI for the nerve agents and / or actual enhancement of its machinery to break down these toxins. Aims 1 and 2 will address the most successful approach to functional expression of various mutant proteins. Based on the results obtained, aims 3 and 4 seek to refine our knowledge of the substrate specificity of this enzyme. We estimate that if the catalytic potential of HuPONI can be enhanced by at least 10-fold, it truly could be a viable anti-nerve agent drug.